Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
  • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/19—Platelets; Megacaryocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/46—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
  • C12N2760/18132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

• “Treatment of Neoplasms with Viruses” (WO 00/62735) relates to a method of administering viruses that are able to replicate and kill neoplastic cells with a deficiency in the IFN-mediated antiviral response.
• One specific aspect of this patent involves the “systemic” administration of such viruses.
• Paramyxoviruses are known to interact with erythrocytes and agglutinate them. They are reported to elute from erythrocytes with lower efficiency than influenza viruses. Howe, C. and Lee, L. T. (Adv. Virus Res. 17:1-50, 1972). Furthermore, the specific inhibition of hemagglutination of erythrocytes caused by paramyxoviruses through neutralization by antibodies specific to viral coat proteins is a well understood phenomenon.
• Bonina et al (Giorn. Batt. Virol. Immun., LXXVIII, 254-261, 1985) show that human macrophages could support the growth of NDV.
• Woodruff et al (Cellular Immunology 5:296-306, 1972) and Woodruff and Woodruff (J of Immunology 112 (6);2176-2183, 1974) found that NDV agglutinates rat, mouse and human lymphocytes in vitro.
• NDV binding of NDV to cells occurs through the interaction of the Neuraminidase of the viral HN protein with sialic acid residues attached to cell surface proteins.
• Human erythrocytes have a very high density of sialic acid residues attached to surface proteins.
• the ratio of erythrocytes to leukocytes in human blood is approximately 1000 to 1.
• NDV binds to the leukocyte fraction instead of the much more numerous erythrocytes.
• This invention provides a method of treating a human subject with cancer, comprising administering to the subject an amount of a pharmaceutical composition effective to treat the subject, the pharmaceutical composition comprising human leukocytes and a replication-competent oncolytic virus in suspension in a physiologically acceptable solution, wherein the virus binds specifically to the leukocytes; and the ratio of plaque-forming units of the virus to number of leukocytes in the composition is at least 1:100, thereby treating the subject.
• This invention provides a method of treating a human subject with cancer, comprising administering to the subject an amount of a pharmaceutical composition effective to treat the subject, the pharmaceutical composition comprising human cells infected with an oncolytic virus in suspension in a physiologically acceptable solution, wherein the cells are leukocytes or platelets, thereby treating the subject.
• This invention provides the use of a pharmaceutical composition to treat a human subject with cancer or in the manufacture of a medicament for the treatment of cancer, the pharmaceutical composition comprising: (a) human leukocytes and a replication-competent oncolytic virus in suspension in a physiologically acceptable solution, wherein the virus binds specifically to the leukocytes and the ratio of plaque forming units of the virus to number of leukocytes in the composition is at least 1:100; or (b) human cells infected with an oncolytic virus in suspension in a physiologically acceptable solution, wherein the cells are leukocytes or platelets.
• Uses (a) and (b) are linked in that practicing use (a) generally involves practicing use (b) since the replication-competent oncolytic virus will generally infect the leukocytes.
• This invention is based, in part, on the finding that an oncolytic virus such as NDV binds to leukocytes and platelets.
• NDV binds leukocytes preferentially compared to erythrocytes. Tumors involve inflammatory processes. Therefore leukocytes to which an oncolytic virus is bound or which are infected with an oncolytic virus are a particularly effective means of delivering oncolytic viruses.
• human cells means cells isolated from a human, or cultured cells that have been derived from cells isolated from a human and/or whose nucleic acid component has been altered by, for example, immortalization, irradiation or recombinant means.
• Human leukocytes and “human platelets” are human cells that are leukocytes and platelets, respectively. Except where otherwise specified or required by the context the terms “cells” or “human cells” refer to leukocytes and/or platelets.
• plaque-forming unit means one infectious virus particle.
• MOI multipleplicity of infection
• clonal virus means a virus derived from a single infectious virus particle and for which individual molecular clones have significant nucleic acid sequence homology.
• sequence homology is such that at least eight individual molecular clones from the population of virions have a sequence homology greater than 95% over 300 contiguous nucleotides.
• LVC leukocyte virus complex
• a virus is said to “bind(s) specifically” to a given cell if such virus binds to such cell with a greater specificity than such virus binds to erythrocytes.
• the terms bind(s) specifically and specifically bind(s) are used interchangeably.
• NDV Newcastle Disease Virus
• replication-competent virus refers to a virus that produces infectious progeny in cancer cells.
• the human cells can be derived from any source. They can be donated by someone other than the subject. However if feasible it is generally preferred to use cells donated by the subject, for safety reasons.
• cells isolated from the donor can first be grown in culture and the cultured cells are also considered to be cells of the donor from which they were derived. Examples of cultured cells that can be utilized in accordance with this invention include immortalized human leukocyte cell lines. Suitable cell lines are publicly available from sources such as the American Type Culture Collection, for example U-937 (ATCC No. CRL-1593.2) and KG-1 (ATCC No. CCL-246).
• the leukocytes utilized in accordance with this invention can be active or inactive.
• Techniques for inactivating leukocytes include irradiation.
• the cells utilized in accordance with this invention can be isolated (for example by leukopheresis in the case of leukocytes). However it is not necessary to isolate the cells and whole blood can be used instead, in which case the pharmaceutical composition comprises the oncolytic virus suspended in whole blood or whole blood containing leukocytes and/or platelets infected with the virus. Optionally the leukocytes or platelets are first isolated from whole blood, mixed or infected with the virus and then added back to the other blood components.
• the leukocytes are selected from monocytes, neutrophils and lymphocytes.
• the leukocytes are tumor-infiltrating lymphocytes (TILs).
• TILs may be prepared for example by the method described in Rabinowich, H., et al., (Cancer Res. 47: 173-7, 1987).
• the oncolytic virus utilized can be of low (lentogenic), moderate (mesogenic) or high (velogenic) virulence.
• the level of virulence is determined in accordance with the Mean Death Time in Eggs (MDT) test. (Alexander, “Chapter 27: Newcastle Disease” in Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3 rd ed., Purchase, et al. eds. (Kendall/Hunt, Iowa), page 117.)
• Viruses are classified by the MDT test as lentogenic (MDT>90 hours); mesogenic (MDT from 60-90 hours); and velogenic (MDT ⁇ 60 hours).
• the virus is a clonal virus.
• the ratio of plaque-forming units of the virus to number of leukocytes in the composition is at least 1:1.
• the leukocytes be saturated with active virus particles.
• NDV saturation is achieved at a 200:1 ratio of plaque-forming units of the virus to number of leukocytes.
• the virus is NDV and the ratio of plaque-forming units of the virus to number of leukocytes in the composition is from about 1:1 to about 200:1, and preferably is about 200:1.
• the pharmaceutical composition utilized comprises cells infected with an oncolytic virus
• the infected cells are at least one-tenth of one percent (0.1%) of the total number of leukocytes and platelets in the composition, more preferably at least thirty percent and most preferably about one hundred percent.
• the virus utilized can be replication incompetent although preferably it is replication competent.
• the oncolytic virus is selected from the group consisting of a Newcastle Disease Virus (NDV), a Mumps Virus, a Measles Virus, a Vesicular Stomatitis Virus, a Para-influenza Virus, an Influenza Virus, an Adenovirus, a Herpes I Virus, a Vaccinia Virus, and a Reovirus.
• NDV Newcastle Disease Virus
• Mumps Virus a Measles Virus
• Vesicular Stomatitis Virus a Para-influenza Virus
• an Influenza Virus an Adenovirus
• Herpes I Virus a Herpes I Virus
• a Vaccinia Virus a Reovirus.
• a Newcastle Disease Virus strain of moderate virulence can be utilized.
• the effective amount is a daily dosage of the composition containing from 6 ⁇ 10 6 to 6 ⁇ 10 10 leukocytes per square meter of patient surface area, for example about 6 ⁇ 10 7 leukocytes per square meter of patient surface area.
• the effective amount is typically a daily dosage of the composition containing from 10 9 to 10 11 platelets per square meter of patient surface area, for example about 10 11 platelets per square meter of patient surface area.
• the daily dosage of the composition can be administered to the subject in multiple administrations in the course of a single twenty-four hour period in which a portion of the daily dosage is administered at each administration. More preferably the daily dosage is administered in a single administration. In an embodiment of this invention the daily dosage of the composition is administered to the subject at a frequency of from one to seven times (i.e. on each of from one to seven days) in a one-week period.
• any conventional route of administration is suitable for administering the pharmaceutical composition.
• the composition can be administered intravenously, intratumorally, intraperitoneally or intravesicularly (kidneys).
• intravenous administration it is convenient if the volume of the composition administered is from twenty-five milliliters to one liter.
• intratumoral administration it is convenient if the volume of composition administered is from one hundred microliters to ten milliliters per tumor mass.
• intraperitoneal administration it is convenient if the volume of composition administered is up to two liters.
• intravesicular administration it is convenient if the volume of composition administered is up to seventy-five milliliters, preferably from fifty to sixty milliliters.
• the concentration of the composition can be varied to achieve the desired volume.
• the composition can be administered by any of the routes given above, for example intravenously or intratumorally.
• the cancer is other than a solid tumor (e.g. leukemia) the composition is not administered intratumorally and instead can be administered by the other routes given above, for example intravenously.
• NDV Newcastle Disease Virus
• the samples were washed twice by adding 2 ml of cold PSB, centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solution away from the cell pellet.
• the PSB was removed each time by aspiration.
• the volumes of each sample were adjusted to 1 ml by adding cold PSB.
• the monoclonal antibody Mab2F12 was added to each sample by adding 20 ul of a solution containing 9.1 ⁇ g of the antibody.
• the samples were incubated for 30 minutes on ice and washed twice again as previously described.
• the goat anti-mouse—PE reporter antibody was added to each sample by adding 1 ml of a 12 ⁇ g/ml solution of this antibody.
• results of the experiment shown in Table 1 indicate that NDV binds preferentially to the leukocyte fraction of human whole blood.
• MOI MOI of 0.05 (to whole blood)
• 46% of the leukocytes are positive for NDV compared to 0% of the erythrocytes.
• MOT MOT of 0.2
• NDV is present on 89% of the leukocytes while bound to only 15% of the erythrocytes.
• MOI's of 0.05 and 0.2 are approximately 50 and 200 to the leukocytes if the presence of the erythrocytes is discounted.
• the small amount of NDV binding to erythrocytes at the higher MOI may reflect low affinity binding to sialic residues on proteins present on the surface of these cells.
• NDV Newcastle disease virus
• the binding of NDV to cells is thought to occur through the interaction of the Neuraminidase activity of the viral HN protein to sialic acid residues attached to cell surface proteins.
• Human erythrocytes have a very high density of sialic acid residues attached to surface proteins for the purpose of keeping the cells in solution in the blood.
• the ratio of erythrocytes to leukocytes in human blood is approximately 1000 to 1, it is especially surprising that NDV binds to the leukocyte fraction instead of the vastly more numerous erythrocytes.
• NDV Associates with Leukocytes in the Presence of NDV Neutralizing Antibody
• Leukocyte bands Two Leukocyte bands (a polymorphonuclear cell band and a mononuclear cell band) were collected and placed in 0.5 ⁇ DMEM. Cells were pelleted and resuspended in full strength DMEM. Cells were washed several times to remove the separation media. Leukocytes in each aliquot were enumerated using a Coulter Counter. Known numbers of leukocytes or volumes of plasma were co-cultured or inoculated onto monolayers of HT1080 human fibrosarcoma cells (ATCC, CCL-121) in 25 cm 2 tissue culture flasks (Corning). HT1080 cells are highly sensitive to cytolysis by NDV. Monolayers were evaluated qualitatively over several days for the presence of CPE (cytopathic effect). Flasks which exhibited CPE were considered positive.
• NDV elicits a humoral immune response which results in the production of neutralizing antibody. Binding of virus to leukocytes may allow the virus to be protected from the neutralizing antibody. This is an advantage over free virus, which is exposed to the neutralizing antibody and rendered non-infectious.
• NDV is not pathogenic in man it is a surprising result to find that NDV binds preferentially to the white blood cell component of human blood.
• Human Leukocytes (5 ⁇ 10 +8 cells) are prepared by leukopheresis or by gradient centrifugation employing POLYMORPHPREP (Nycomed, Inc.) using the manufacturer's instructions. The cells are washed twice in sterile 1 ⁇ PBS at room temperature and brought to a volume of 10 ml in the same buffer. The cells are mixed with 1 ⁇ 10 +10 pfu of NDV (added aseptically) and allowed to sit for 30 minutes (with further brief mixings at 10 and 20 minutes). The cells are centrifuged for 5 minutes at 1500 rpm and the PBS removed. The cells are washed with 20 ml of 1 ⁇ PBS and centrifuged again. The cell pellet is diluted to 10 ml for injection.
• Athymic mice are injected intradermally with 10 million human tumor cells. After tumors reached a size range of between 5 and 10 mm, a single injection of 8 ⁇ 10 +6 cells of the Leukocyte/NDV complex described in Example 3 is given. The effect of LVC on tumor growth is examined for complete and partial regressions.
• LVC leukocyte/NDV complex
• LVC leukocyte/NDV complex
• Mouse whole blood was collected in citrate tubes (3.2%, 0.105M, Becton Dickinson #366415). Cells were counted using the trypan blue exclusion method and a hemocytometer. NDV lot number RL-005 (4.2E+10 PFU/ml) was used to infect the cells at MOIs (Multiplicity of Infection, expressed as PFU/cell) of 0.2, 1 and 3 (along with a negative control of no added virus). After virus was added, tubes were incubated at 37° C. for 30 minutes. Gentle mixing of the tubes to keep cells in suspension was performed at 3 intervals during the incubation period.
• MOIs Multiplicity of Infection, expressed as PFU/cell
• the samples were washed twice by adding 2 ml of cold PSB, centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solution away from the cell pellet.
• the PSB was removed each time by aspiration.
• the volumes of each sample were adjusted to 1 ml by adding cold PSB.
• the monoclonal antibody Mab2F12 was added to each sample by adding 20 ul of a solution containing 9.1 ⁇ g of the antibody.
• the samples were incubated for 30 minutes on ice and washed twice again as previously described.
• a goat anti-mouse—PE reporter antibody was added to each sample by adding 1 ml of a 12 ⁇ g/ml solution of this antibody.
• results The results of the experiment shown in Table 4 indicate that unlike the binding of NDV to human blood cells where the virus preferentially binds to leukocytes (Example 1), NDV binds preferentially to the erythrocyte fraction of whole mouse blood and does not bind to the leukocytes.
• mouse leukocytes are negative.
• the samples were washed twice by adding 2 ml of cold PSB, centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solution away from the cell pellet.
• the PSB was removed each time by aspiration.
• the volumes of each sample were adjusted to 1 ml by adding cold PSB.
• the monoclonal antibody Mab2F12 was added to each sample by adding 20 ul of a solution containing 9.1 ⁇ g of the antibody.
• the samples were incubated for 30 minutes on ice and washed twice again as previously described.
• a goat anti-mouse—PE reporter antibody was added to each sample by adding 1 ml of a 12 ⁇ g/ml solution of this antibody.
• results The results of the experiment shown in Table 5 indicate that NDV binds to rat leukocytes at a low MOI (0.02 and 0.05) while it does not bind well to the erythrocytes at these MOI's.
• the pattern appears to be intermediate between the binding of the virus to human leukocytes to which it binds preferentially and to mouse leukocytes, to which it does not bind.
• the samples were washed twice by adding 2 ml of cold PSB, centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solution away from the cell pellet.
• the PSB was removed each time by aspiration.
• the volumes of each sample were adjusted to 1 ml by adding cold PSB.
• the monoclonal antibody Mab2F12 was added to each sample by adding 20 ul of a solution containing 9.1 ⁇ g of the antibody.
• the samples were incubated for 30 minutes on ice and washed twice again as previously described.
• a goat anti-mouse—PE reporter antibody was added to each sample by adding 0.1 ml of a 12 ⁇ g/ml solution of this antibody.
• Results The results of the experiment shown in Table 6 indicate that NDV binds to human platelets.
• the number of platelets that are positive for binding of NDV does not increase greatly in the MOI range tested although the mean fluorescence does, indicating that more NDV binds to the positive portion of the platelets as the MOI increases.
• the samples were washed twice by adding 2 ml of cold PSB, centrifuging for 5 minutes, 4° C., 2000 rpm, and aspirating the solution away from the pellet. The volumes of each sample were adjusted to 100 ⁇ l with PSB. Monoclonal antibody Mab2F12 was added to each sample by adding 2 ⁇ l of a solution made in PSB containing 1 ⁇ g of the antibody. The samples were incubated for 30 minutes on ice and washed twice again as previously described. The goat anti-mouse—PE reporter antibody was added to each sample by adding 100 ⁇ l of a 12 ⁇ g/ml solution of this antibody diluted 2.4:1 in PSB. The samples were incubated for 30 minutes on ice and washed as described above.
• each sample was incubated with 3 ml of 1 ⁇ FACS Lysing Solution for 6 minutes at room temperature, then centrifuged and aspirated as before. The cell pellets were re-suspended in 0.5 ml of PSB. Sample analysis was performed with a Becton Dickinson FACSCaliburTM flow cytometer. Granulocyte, lymphocyte and monocyte populations were gated by comparing the forward scatter and side scatter parameters for each sample. This was used to determine the number of cells positive for binding the virus and the mean fluorescence value of each of these populations for each sample.
• the results (Table 7) indicate that NDV binds preferentially to the three cell populations.
• the order of preference is: monocyte>granulocyte>>lymphocyte.
• the monocyte population binds substantially more NDV than does the granulocyte population, and binds much more virus than does the lymphocyte cell population.

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